Bradykinin is a hormonal nonapeptide (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg)(SEQ ID NO:1) which mediates pain, vascular permeability, inflammation, gastrointestinal function, and smooth muscle tone in vascular and other tissues. Bradykinin (BK) is one of the key mediators of the body""s response to trauma and injury. BK levels are generally low until a traumatic event triggers a cascade of biochemical reactions and a rise in the concentration of BK by proteolytic generation. High molecular weight precursors, the kininogens, are found in blood and tissue. This cascade is initiated by the activation of the Hageman factor which also initiates fibrinolysis and coagulation.
Receptors for BK exist in the nervous system, epithelia, smooth muscle and fibroblasts. In each tissue type BK triggers specific responses including neurotransmitter release, muscle contraction, fluid secretion by epithelia, and the stimulation of cell growth. It can also act as a neurotransmitter.
The initial interaction for biological response occurs at a BK receptor site on a cell. Specific BK antagonists have been developed (Vavrek, Peptides, 6, 161-165 (1985)). Their potential use includes use as anti-nociceptive and anti-inflammatory agents. Bradykinin activates neurons and produces neurotransmitter release. It also stimulates the production of a number of bioactive intermediates including inositol triphosphate (Ins-1,4,5-P3) and diacylglycerol (DAG) and arachidonic acid (AA) and its cyclooxygenase and lipooxygenase products. These substances cause cellular levels of cAMP, cGMP, and Ca2+ to increase. BK also activates phospholipase C and A2. In neurons, the most important points of action for the substances released by BK stimulation are ion channels. Miller, R. J., Trends Neurosci., 10, 226-228 (1987).
Bradykinin released during tissue damage causes vasodilation, increased vascular permeability, altered gut motility and pain. Specific bradykinin receptors exist in intestinal mucosa and muscle. Bradykinin and analogues stimulate C1 secretion in the gut. Specific BK receptor binding sites occur in the mucosa and in muscle. BK has a contractile effect in muscle. Manning et al., Nature, 299, 256-259 (1982).
Addition of nanomolar concentrations of BK to the serosal surface of the mucosal later of the guinea pig ileum rapidly increased transepithelial potential difference (p.d.) and the short circuit current (ISC). This suggests localization of BK receptors at the serosal surface of the villus and crypt epithelium. The increase in ISC is thought to be due to stimulation of anion secretion (C1 out of the cell produces a larger potential difference). Manning et al., Nature, 299, 256-259 (1982).
Bradykinin could open calcium channels as indicated by the inhibitory effects of Ca2+ channel blockers. Calcium may be involved in regulating BK receptor binding. See Innis et al., Proc. Natn. Acad. Sci., 2630-2634 (1981). BK also stimulates sodium intake and DNA synthesis. Owen et al., Cell, 32, 979-985 (1983).
Excessive kinin activity may play some role in carcinoid syndrome and in inflammatory bowel disease. Patients with ulcerative colitis have abnormally high levels of active kallikrein, the kinin-releasing enzyme and plasma and tissue levels of peptidiyl dipeptidase which degrades kinins are depressed in patients with regional enteritis. Manning et al., Nature, 299, 256-259 (1982).
Autoradiographic studies localize BK receptor binding sites to the substantia gelatinosa, dorsal root, and a subset of small cells in both the dorsal root and trigeminal ganglia of the guinea pig. Binding was also observed over myocardial/coronary visceral afferent fibers. The localization of BK receptors to nociceptive pathways supports a role for BK in pain mediation. Several BK antagonists block BK induced acute vascular pain in the rat. BK antagonists also relieve BK and urate induced hyperalgesia in the rat paw. These results indicate that BK is a physiologic mediator of pain and that BK antagonists have analgesic activity in both acute and chronic pain models. The BK receptor involved in vascular pain may be different from the receptor involved in cutaneous hyperalgesia. Steranka et al., Proc. Natl. Acad. Sci. USA., 85, 3245-3249 (1988).
BK receptors have been classified as two major subtypesxe2x80x94B1 and B2. The BK metabolite des-Arg-bradykinin is a B1 receptor agonist which has higher potency than BK but it is inactive at B2 receptors. Steranka et al., Proc. Natl. Acad. Sci. USA., 85, 3245-3249 (1988). BK also binds to G protein-coupled receptors that activate phospholipase C or phospholipase A2 and increases synthesis of inositol triphosphate or arachidonic acid. Olsen et al., J. Bio. Chem. 263, 18030-18035 (1988). G-proteins are a family of membrane proteins that become activated only after binding guanosine triphosphate (GTP). Activated G-proteins in turn activate an amplifier enzyme on the inner face of a membrane; the enzyme then converts precursor molecules into second messengers. For example, an external signal molecule (bradykinin) may bind to its cell-surface receptor (BK-2) and induce a conformational change in the receptor. This change is transmitted through the cell membrane to a G-protein, making it able to bind to GTP. Binding of GTP causes another conformational change in the G-protein that enables it to activate adenylate cyclase (amplifier enzyme) to initiate formation of cAMP (second messenger).
In Swiss 3T3 fibroblasts, BK stimulated phospholipase C mediated InsP formation and PGE-2 synthesis. G proteins were implicated in the mediation of the effects of bradykinin suggesting that the receptor is bound to a G protein which interacts with the particular enzyme. Burch et al., Proc. Natl. Aca. Sci. USA, 84, 6374-6377 (1987). Two different G-proteins mediate neuropeptide Y and bradykinin stimulated phospholipid breakdown in cultured rat sensory neurons. Perney et al., J Biol. Chem., 264, 7371-7327 (1991).
It is known that there is a large degree of heterogenicity within the muscarinic, adrenergic, and serotonergic class of receptors. Furthermore, xe2x80x9c[s]imple classification of subtypes of BK receptors cannot fully account for the properties of these receptors on cells from a variety of tissues.xe2x80x9d Mahan et al., Mol. Pharmacol., 37, 785-789 (1990).
Bradykinin induced increases in InsP formation through the activation of phosphatidylinositol-specific phospholipase C and subsequent mobilization of intracellular Ca2+ and direct activation of phospholipase A2, which causes the release of arachidonate and subsequent synthesis of prostaglandin E2 have been found to exist in Swiss albino mouse 3T3 cells and BALBc (SV-T2) mouse 3T3 cells and involve receptors coupled to pertussis toxin-insensitive G proteins. These receptors belong to the B2 subtype. Mahan et al., Mol. Pharmacol., 37, 785-789 (1990).
The effect of bradykinin on the neuroeffector junction of the isolated rat vas deferens has been studied. Llona et al., J. Pharmacol. Exp. Ther., 241, 608-614 (1987). BK potentiated the magnitude of the muscular response to the electrically driven twitches and contracted the smooth muscle generating an increased muscle tone. The former action is referred to as the neurogenic or presynaptic effect and the latter is called the musculotropic or postjunctional action. The rat vas deferens contains bradykinin receptors on the nerve endings and on the smooth muscle membrane. The structural prerequisites for the activation of these receptor sites appear to be slightly different. Their results support the existence of B2 receptors. des-Arg9-BK and des-Arg9-[Leu8]-BK are inactive in causing either pre- or postsynaptic BK like responses and incubation of des-Arg-9-[Leu8]-BK at high concentrations failed to antagonize BK responses in the vas deferens. This peptide is a known B1 antagonist. The authors suggest that there are several classes of BK-2 receptors. Llona et al., J. Pharmacol. Exp. Ther., 241, 613 (1987). See also Brass et al., Br. J. Pharmacol., 94, 3-5 (1988).
As indicated, BK mediates vasodilation, pain and smooth muscle contraction in a number of tissues. Many of these biological actions may result from the release of arachidonic acid and its metabolites. The major metabolite in Swiss 3T3 cells (fibroblasts) is PGE2 which induces smooth muscle contraction, mitogenesis, an increase in intracellular free calcium and stimulates adenylate cyclase (to produce cAMP). BK activates phospholipases which control intracellular arachidonate. Conklin et al., J. Pharmacol. Exp. Ther., 244, 646-649 (1988).
Phospholipases are considered to be the rate limiting enzymes in receptor mediated arachidonate release. BK activates PLA2, a phospholipase which cleaves arachidonic acid directly from the parent phospholipid. In contrast, BK in CPAE cells (bovine pulmonary artery endothelial cells) stimulates activity of a phosphatidylcholine-specific PLC which provides arachidonate substrate for PGI2 synthesis. The authors conclude that the BK receptors are pharmacologically distinct and that more BK subtypes exist beyond BK1 and BK2. Conklin et al., J. Pharmacol. Exp. Ther., 244, 646-649 (1988).
To further clarify the role of bradykinin, kinins are released in response to tissue injury and activate sensory pain fibers, contract venous smooth muscle and stimulate prostacyclin (PGI2) synthesis and endothelium derived relaxing factor (EDRF). Blood flow to the damaged area and vascular permeability increase to cause inflammation. Plevin et al., Trends Pharmacol. Sci., 9, 387-389 (1988). Multiple B2 BK receptors in mammalian tissues are present. The tissues include guinea-pig ileum, vas deferens prejunctional, N1E-115 P1 response (neuronal cell line), Rat uterus, and guinea-pig trachea (endothelial cells-BK linked to second messenger and coupled to a G-protein).
Because of the potential molecular heterogenicity of bradykinin receptors in cells and discrepancies in their pharmacological classification, there is a need to elucidate and fully characterize a homogeneous human bradykinin receptor and to express this receptor to measure antagonist or agonist response or interaction.
It is known that cDNAs for a number of receptors of the G protein-coupled superfamily have been cloned. These include, for example, a beta-adrenergic receptor, a substance P receptor, and a neurotensin receptor. Strader et al., Nature 321, 75-79 (1986); Yokata et al., J. Biol. Chem. 264, 17649-17652 (1989); Tanaka et al., Neuron 4, 847-854 (1990).
The present invention is a cloned human BK-2 bradykinin receptor cloned from a human lung fibroblast cell line. A cDNA clone, also part of the instant invention, encodes a novel 364 amino acid protein (BK-2 receptor) that has the characteristics of a seven transmembrane domain G-protein coupled receptor. The present invention is directed to a protein having an activity equal to that of human bradykinin BK-2 receptor wherein this protein is free of other human receptor proteins or substantially free of other human receptor proteins.
This invention claims a human bradykinin BK-2 receptor protein which is free of other human proteins and is a recombinantly produced receptor derived from human cells. This invention claims a human bradykinin receptor protein comprising 364 amino acids with the particular amino acid sequence of (SEQ ID NO: 2).
This invention also is directed to a pharmaceutical composition for inhibiting the binding of bradykinin to human bradykinin BK-2 receptor wherein the composition comprises an effective amount of bradykinin BK-2 receptor. It is further directed to a method of inhibiting the binding of bradykinin to human bradykinin BK-2 receptor, in a patient in need of such inhibition, comprising administration of an effective amount of bradykinin BK-2 receptor.
This invention is also directed to a DNA sequence encoding human bradykinin BK-2 receptor wherein the sequence is free of other human DNA sequences. This invention claims an open reading frame coding for the human BK-2 bradykinin receptor protein with the DNA sequence (SEQ ID NO:3) or a degenerate variation thereof. This invention further claims a DNA sequence comprising the sequence (SEQ ID NO:4) or a degenerate variation thereof. This invention further comprises a DNA sequence (SEQ ID NO:5) or a degenerate variation thereof.
This invention comprises a DNA sequence which is SEQ ID NO: 6 or a degenerate variation thereof. This invention is directed to a DNA sequence which is (SEQ ID NO:4) or a degenerate variation thereof.
Also claimed is an oligonucleotide probe that is capable of hybridizing with DNA that encodes the bradykinin BK-2 receptor and which is labeled with a detectable moiety. The oligonucleotide probe is of the sequence (SEQ ID NO: 7) or a degenerate variation thereof.
This invention is directed to an expression construct, which comprises a mammalian cell vector, and the base sequence encoding human bradykinin BK-2 receptor protein. This expression construct may be pCDNAI-Neo, and the base sequence encoding human bradykinin BK-2 receptor protein. The expression construct contains a DNA sequence which comprises the sequence (SEQ ID NO: 3) or a degenerate variation thereof. The expression construct further comprises the sequence which is (SEQ ID NO: 4) or a degenerate variation thereof. The expression construct further comprises the sequence which is (SEQ ID NO: 5) or a degenerate variation thereof.
This invention claims COS cells or Chinese Hamster Ovarian (CHO) cells transfected with an expression construct as defined above. Other mammalian cells or cell lines may also be treated with an expression construct including plasmids which contain a recombinant DNA sequence that codes for the human BK-2 bradykinin receptor protein. The expression construct or the transfected cell line may contain the necessary promoter sequence and the transcriptional and translational proteins or biomolecules necessary to express the human bradykinin receptor protein in the selected cell line.
This invention also claims a method of using a COS-7 cell line, said line transfected with an expression construct wherein the expression construct comprises a mammalian expression vector, and the base sequence encoding human bradykinin BK-2 receptor protein, comprising the steps of: expressing cloned human bradykinin BK-2 receptor in the COS-7 cells; incubating radiolabeled bradykinin and an optional test compound with the expressed human bradykinin BK-2 receptor to form a radiolabeled bradykinin-receptor complex or a test compound-receptor complex; separating said radiolabeled-receptor complex or said test compound-receptor complex from unbound radiolabeled bradykinin; measuring the amount of said radiolabeled-receptor complex. A mammalian expression vector used in the present invention is pcDNA I-Neo.
This invention is also directed to a CHO cell line wherein the cell line is transfected with an expression construct containing the cloned human BK-2 bradykinin receptor gene. It is also directed to a method of using a Chinese hamster ovarian cell line wherein the cell line is transfected with an expression construct and the expression construct comprises a mammalian expression vector, and the base sequence encoding human bradykinin BK-2 receptor protein, comprising the steps of: expressing cloned human bradykinin BK-2 receptor in the Chinese hamster ovarian cells; incubating radiolabeled bradykinin and an optional test compound with the expressed human bradykinin BK-2 receptor to form a radiolabeled bradykinin-receptor complex or a test compound-receptor complex; separating the radiolabeled-receptor complex or the test compound-receptor complex from unbound radiolabeled bradykinin; and measuring the amount of the radiolabeled-receptor complex. The mammalian expression vector used in the above method is pcDNA I-Neo.
This invention also claims a method of using a Chinese hamster ovarian cell line, said line transfected with an expression construct wherein the expression construct comprises a mammalian expression vector, and the base sequence encoding human bradykinin BK-2 receptor protein, comprising the steps of: expressing cloned human bradykinin BK-2 receptor in the Chinese hamster ovarian cells; equilibrating the expressed human bradykinin receptor with fura-2 to incorporate the fura-2 into the Chinese hamster ovarian cell; washing the Chinese hamster ovarian cell to remove unassociated fura-2; incubating the washed Chinese hamster ovarian cell with bradykinin and an optional test compound to induce an intracellular Ca2+ release; photometrically measuring the intracellular Ca2+ release. The mammalian expression vector used in the above method is pcDNA INeo.
The present invention is a cloned human BK-2 bradykinin receptor protein and DNA sequence that codes for this protein receptor. It is known that a rat BK-2 bradykinin receptor has been cloned and expressed in Xenopus laevis oocytes. See McEachern et al., Proc. Natl. Acad. Sci. USA 88, 7724-7728 (1991). In this reference, the authors describe the isolation of a cDNA encoding a functional smooth muscle bradykinin BK-2 receptor from a rat uterus library by a clonal selection strategy. This cDNA was expressed in Xenopus laevis oocytes and assayed for bradykinin responses. The predicted protein is homologous to the seven transmembrane G protein-coupled superfamily of receptors.
The present invention, however, is directed to the isolation and characterization of a cloned human BK-2 bradykinin receptor protein and is therefore critical for assisting in the discovery of therapeutic compounds that act as antagonists or as an agonist of the human BK-2 receptors.
This invention describes the cloning and expression of a human bradykinin BK-2 receptor and is therefore critical for drug antagonist or agonist studies and for eventual treatment of disorders or diseases associated with bradykinin elicited responses. Previously, rat or guinea pig tissues were utilized to screen for BK antagonists. The cloned human BK receptor circumvents the problem of species variability and therefore is valuable for human agonist or antagonist studies. The cloned DNA claimed in the instant invention provides a significant advantage over human cell lines containing the BK receptor in antagonist or agonist studies because of the potential problem of various receptor subtypes in a given cell line. Expression of the human BK-2 receptor in a cell line lacking any endogenous BK receptors alleviates this problem. Advantageously, expression of the cloned human BK-2 receptor in a cell line that lacks endogenous BK receptors permits the identification of compounds that specifically interact with this receptor. In a preferred embodiment, the cloned human BK-2 receptor will be introduced into a stable mammalian expression system which will be used to screen for antagonists of the BK-2 receptor.
This invention concerns a human bradykinin BK-2 receptor protein. (SEQ. ID NO: 2).
This receptor protein comprises a 364 amino acid sequence wherein the protein is substantially free of other human receptor proteins. This invention also encompasses mutated proteins which have substantially similar binding activities to the cloned and expressed protein claimed in this invention.
A pharmaceutical composition containing human bradykinin receptor may be used to inhibit the binding of bradykinin to cellular bradykinin BK-2 receptor wherein the composition contains an effective amount of bradykinin BK-2 receptor. This invention further concerns a method of inhibiting the binding of bradykinin to cellular human bradykinin receptor, in a patient in need of such inhibition comprising administration of an effective amount of human BK-2 receptor.
This invention is also directed to a DNA sequence encoding human bradykinin BK-2 receptor complementary DNA wherein this DNA is free of other human DNA sequences: (SEQ. ID NO: 6).
It is also well known, that there is a substantial amount of redundancy in the various codons which code for specific amino acids. Therefore, this invention is also directed to those DNA sequences which contain alternative codons which code for the eventual translation of the identical amino acid. For purposes of this specification, a sequence bearing one or more replaced codons will be defined as a degenerate variation. Also included within the scope of this invention are mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
This invention is directed to a complementary DNA sequence encoding a full length BK-2 receptor beginning with a codon at nucleotide 136 and ending at the in-frame translation termination codon 1315 (SEQ ID NO: 3) This invention is further directed to a partial cDNA derived from human uterus of 386 nucleotides which encompasses nucleotides 540 to 926 of the gene for the full length BK-2 receptor and is used as a probe to screen a cDNA library prepared from a human fibroblastic lung cell line (SEQ ID NO: 7). In addition, this invention describes and claims two positive clones CCD-16-2 (0.78 kb) (SEQ ID NO: 8) and CCD-16-6 (1.1 kb) (SEQ ID NO: 9).
This invention also concerns and claims systems for expressing a human bradykinin BK-2 receptor. This system includes mammalian expression vectors which incorporate a base sequence encoding human bradykinin BK-2 receptor protein. This expression vector is then used to transfect a suitable expression host which translates the genetic information, synthesizes the protein or partial protein, and enables expression of the cloned BK-2 receptor protein.
This invention further claims a method of using an expression system containing a cloned human bradykinin BK-2 receptor to determine the binding affinity of bradykinin, bradykinin receptor antagonists, a bradykinin receptor agonist, or bradykinin analogues. A suitable vector containing a cDNA of human bradykinin BK-2 receptor is prepared according to the methods described in the instant invention, transfected into an appropriate host system, such as COS-7 or CHO, expressing a BK-2 bradykinin receptor protein, and contacting the described system with labeled bradykinin. After a period of incubation, the cellular mixture is filtered to separate bound material from unbound material with the radiolabeled and bound material (receptor plus bradykinin) quantified by liquid scintillation counting or other suitable means. This method further comprises competitive binding assays using bradykinin receptor antagonists, agonists, and analogues. This method comprises the steps of:
(1) expressing human BK-2 receptor in a suitable expression host such as COS-7 or CHO;
(2) treating the human BK-2 receptor with radiolabeled bradykinin while simaltaneously treating the human BK-2 receptor with a test compound to form a receptor-ligand complex;
(3) separating radiolabeled receptor-ligand complex from unbound radiolabeled bradykinin;
(4) measuring the radioactivity of bound radiolabeled bradykinin.
Alternatively, other suitable binding assays may be used to detect and moniter the activity of an expressed bradykinin receptor. For example, in CHO cells the release of intracellular calcium stores in response to the binding of bradykinin to the expressed bradykinin BK-2 receptor may be monitered by voltage means or by chemical dye means.
A human BK-2 bradykinin receptor is cloned from the lung fibroblast cell line CCD-16Lu. The cDNA clone (SEQ ID NO: 6) encodes a 364 amino acid protein (SEQ ID NO: 2) that has the characteristics of a seven transmembrane domain G-protein coupled receptor. The predicted amino acid sequence of the human BK-2 receptor is approximately twenty percent different than the protein isolated from the smooth muscle rat BK-2 receptor (81% homologous). McEachern et al., Proc. Natl. Acad. Sci. USA 88, 7726 (1991). Transfection of the human BK-2 receptor cDNA into COS-7 cells results in the expression of high levels of specific BK binding sites. Saturation binding analysis indicates that the human BK-2 receptor expressed in COS-7 cells binds BK with a KD of 0.13 nM. Pharmacological characterization of the expressed BK receptor cells demonstrates and is consistent with a cDNA encoding for a BK-2 receptor subtype.
The particular cDNA claimed and disclosed in the instant invention is prepared, isolated, and expressed by combining reverse PCR techniques (to generate a probe) with screening methods to detect the cDNA which codes for a functional BK-2 receptor. Reverse PCR with Thermus thermophilus DNA polymerase (PERKIN ELMER CETUS, Norwalk Conn.) is performed using human uterine mRNA (CLONETECH, Palo Alto, Calif.). Annealing of the reverse primer and reverse transcription is done by incubating the PCR reaction minus the forward primer for 10 minutes each at room temperature, 42xc2x0 C., and 60xc2x0 C. Two rounds of PCR are performed using degenerate primers with the restriction site adapters, NotI on the forward primer CGGCGGCCGCGCNAAYAAYTTYGAYTGG (SEQ ID NO: 10) and XhoI on the reverse primer CGCTCGAGCGYTTYTTYTTYTCNGTYTG (SEQ ID NO: 11). The degenerate primers are designed using the hypothesized rat amino acid sequence of the rat BK-2 bradykinin receptor. These primers are removed using a CENTROCON 30 (AMICON, Beverly, Mass.) and a third round of PCR is performed using a second pair of nested primers (with restriction site adapters) GCGCGGCCGCAAYACNATGATHTA (SEQ ID NO: 12) and CGCTCGAGACYTCYTTRAAYTTYTTCAT (SEQ ID NO: 13). PCR products are then analyzed on a 3.5% NUSIEVE (FMC BIOPRODUCTS, Rockland, Me.) gel. A 386 bp PCR product is then subcloned into pBLUESCRIPT (STRATGENE, La Jolla, Calif.) and characterized by DNA sequence analysis. This PCR product is then used as the model for the labeled probe necessary to screen a cDNA library to detect a cDNA that codes for a BK-2 bradykinin receptor. Use of PCR to generate probes specific for uncloned genes is known. See Sambrook et al., Molecular Cloning, Volume 2, 14.7, Cold Spring Harbor Lab. Press (1989). In general, mRNA (or fragments thereof) is extracted from cells which contain the target protein and is used as a template for construction of cDNA using reverse transcriptase. This cDNA is then used as a template for degenerate pools of oligonucleotide primers and for the resultant PCR amplification products. These products are cloned into an appropriate vector, sequenced, and then used as a probe to screen a cDNA library.
Isolation of cDNA coding for the human BK-2 bradykinin receptor is accomplished by first, isolating mRNA from the human cell line CCD-16Lu (CCL 204 obtained from the ATCC, Rockville, Md.) using the INVITROGEN FAST TRACK system (Invitrogen, San Diego, Calif.) according to the manufacturer""s instructions. cDNA is then prepared from mRNA using the BRL cDNA Synthesis System (BRL, Gaithersburg, Md.). See Gubler et al., Gene 25, 263 (1983) as modified by BRL. BstXI adapters are then added and the modified cDNA ligated into pcDNA II (INVITROGEN). Bacterial colonies are then plated at a density of 30,000 colonies per plate and transferred to duplicate Durulose-UV (STRATAGENE) filters using standard techniques (MANIATIS). The probe utilized for screening is generated by random primed synthesis (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) of the 386 bp PCR product described above in the presence of [alpha-32P]dCTP (NEN, Boston, Mass.). Duplicate filters are also hybridized with 1.5xc3x97106 cpm/ml [32P] labeled probe in 50% formamide hybridization solution, [5xc3x97SSC, 5xc3x97Denhart""s, 100 ug/ml DNA, (SIGMA, St Louis, Mo.)] at 50xc2x0 C. for 12 hours. The filters are washed at high stringency in a final wash of 0.1xc3x97SSC, 0.1% SDS at 60xc2x0 C. Positive colonies are then rescreened as before. Plasmid is then isolated from second round positives and the DNA sequence is determined by double strand DNA sequencing using the SANGER METHOD and SEQUENASE (US BIOCHEMICALS, Cleveland, Ohio).
In order to enable expression of the cDNA that encodes for the BK-2 bradykinin receptor, COS-7 cells are transfected using LIPOFECTIN (BRL, Gaithersburg, Md.) with 50 ug/107 cells of the BK-2 receptor cDNA subcloned into the eucaryotic expression vector pcDNA I-Neo (Invitrogen). Cells are then harvested after 72 hours and the membranes containing the expressed receptor protein are prepared by scraping the cells in phosphate buffered saline solution and centrifuging for ten minutes at 500xc3x97g. The cell pellet is then resuspended and homogenized with a Polytron in 20 mM N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES, pH 6.8 at room temperature) buffer containing 1 mM 1,10 phenanthroline. The homogenate is then centrifuged for ten minutes at 500xc3x97g. The final membrane pellet is resuspended in assay buffer (TES plus 0.1% protease free bovine serum albumin, 5 uM MK-422 (enalaprilat; Gross et al., J. Pharmacol. and Exper. Ther. 216, 552-557 (1981)) and 140 ug/ml bacitracin using a motor-driven teflon-glass tissue homogenizer. Protein determination are performed by the method of Bradford using bovine IgG as the standard. See Bradford, M. A. Anal. Biochem. 72, 248-254 (1976).
Binding assays are then performed to determine receptor antagonist or agonist interaction. The assays utilized in the instant invention follow the method of Manning et al., J. Pharmacol. Exp. Ther., 237, 504-512 (1986). [3H]BK at various concentrations is incubated for 60 minutes at 25xc2x0 C. with approximately 50 ug membrane protein in a volume of 1 ml. The assay is terminated by filtration over Whatman GF/B filters presoaked for 3 hours in 0.1% polyethyleneimine using a BRANDEL M-24 CELL HARVESTER (BRANDEL, Gaithersburg, Md.). The tubes are then rinsed two times with 4 ml ice-cold 10 uM TES and the filter bound radioactivity is quantitated by liquid scintillation counting. Nonspecific binding is determined by performing incubation in the presence of 1 uM BK and generally represents less than 5% of the total binding at 100 pM [3H]BK. Competition binding experiments, in the presence of 100 pM[3H]BK, are performed with varying concentrations of the test compound(s). The competition and saturation experiments are analyzed using the EBDA program of McPherson. See McPherson, G. A. J Pharmacol. Methods 14, 213-218 (1985).
As indicated above, the present invention is directed to a novel human bradykinin receptor and cDNA clone encoding this receptor. This cDNA clone is isolated by a combined approach using PCR technology to generate a suitable probe that is then used to screen a cDNA library. Reverse PCR from human uterus mRNA using degenerate primers based on the amino acid sequence of the rat BK-2 receptor (McEachern et al.) is first used to obtain a 386 nucleotide partial cDNA for a human BK-2 receptor. This partial cDNA sequence (SEQ ID NO: 7)encompasses nucleotides 540 to 926 and is 87% identical to the corresponding region of the rat BK-2 cDNA (nucleotides 703 to 1089) (Data Not Shown). The 386 nucleotide partial cDNA is then used to screen, by nucleic acid hybridization, a cDNA library prepared from CCD-16Lu cells. CCD-16Lu is a human fibroblast lung cell line that contains 20,000-30,000 BK receptors per cell and is therefore particularly useful for obtaining the necessary mRNA. Two positive clones, CCD-16-2 (0.78 kb) and CCD-16-6 (1.1 kb) may be isolated from the CCD-16Lu library and subsequently characterized by DNA sequence analysis.
The DNA sequence of clone CCD-16-2 indicates that this clone beings in the 5xe2x80x2 untranslated region of the BK-2 receptor cDNA and ends in the middle of the coding sequence (SEQ. ID NO: 8).
The second clone, CCD-16-6, begins in the coding region and contains an in-frame translation termination codon (SEQ. ID NO: 9)
These two clones overlap for 312 nucleotides and are 100% identical in the overlap region. In addition, both clones are 100% identical in the region spanned by the probe derived from human uterine mRNA. These results indicate that these clones are derived from the same mRNA transcription unit and this transcript is also present in human uterus.
A unique restriction site (SacI or GAGCT!C) in the overlap region of CCD-16-2 and CCD-16-6 permitted the construction of a cDNA clone encoding a full length BK-2 receptor from the cleavage fragments of the two clones (SEQ ID NO: 6). The human BK-2 receptor cDNA clone contains an open reading frame from nucleotide 136-1314 (SEQ ID NO: 3). The initiator methionine codon is believed to be at position 223 Seq. ID No., which is analogous to the proposed initiator methionine in the rat BK-2 cDNA (McEachern et al.). Although two in-frame methionine codons, at nucleotides 142 and 166, occur upstream of the proposed initiator methionine, only the methionine codon at nucleotide 223 contains the elements described by Kozak that are required for efficient initiation of translation. See Kozak, M. J. Cell Biol., 108, 229-241 (1989). The in-frame translation termination codon at nucleotide 1315 is in the analogous position to the termination codon in the rat BK-2 receptor cDNA. The predicted size of the human BK-2 receptor is 364 amino acids corresponding to a weight of 41,140 Daltons.
The human BK-2 bradykinin receptor claimed in the instant invention has an overall amino acid similarity to the rat BK-2 receptor of 87% but also contains striking differences. One significant distinction between the human and rat BK-2 receptor proteins occurs in the N-terminal extracellular region of the respective proteins. The claimed human receptor has two amino acids deleted from the N-terminal extracellular region of the protein receptor while the rat BK-2 receptor retains the two amino acids. This distinction may be critical in determining and distinguishing binding characteristics of bradykinin and various antagonists or agonists to the human, versus the rat, receptor. The three potential sites of N-glycosylation seen in the rat BK-2 receptor (2 in the N-terminal domain and one in the putative extracellular loop between transmembrane helices 4 and 5) are all conserved in the human receptor. The human BK-2 receptor contains several consensus sites for phosphorylation by cAMP dependent protein kinase and protein kinase C in the third intracellular loop and in the carboxy terminal tail. In the beta-adrenergic receptor these regions appear to be involved in receptor coupling to G-proteins, and phosphorylation at analogous sites occurs during receptor desensitization. See Strader et al., FASEF 3, 1825-1832 (1989) and Dohlman, et al., Ann. Rev. Biochem. 60, 653-688. Phosphorylation of these intracellular Ser and Thr residues may affect the ability of the BK receptor to communicate with G-proteins. The highest degree of overall identity seen between the claimed human BK-2 receptor and known other proteins is with the rat angiotensin receptor (32%).
Functional expression of the human BK-2 receptor is obtained by placing the entire BK-2 clone under the control of the CMV promoter (Human cytomeyalo virus) in the eucaryotic expression vector, pCDNAI-Neo (Invitrogen, San Diego, Calif.). This construct is then transfected into COS-7 cells or CHO cells or cell lines and membranes from these cells are analyzed for expression of the BK-2 receptor. Membranes prepared from transfected cells contain specific BK binding sites with a KD of 0.13+/xe2x88x920.09 nM as determined by saturation binding analysis (Data not shown). The level of expressed receptor ranges from 210 to 450 fmole/mg protein. Scatchard analysis of the saturation binding data suggests that there are two classes of BK binding sites on the membrane, a high affinity site (KD=0.13 nM) and a lower affinity site that is not well defined by saturation analysis (KD=3 nMxe2x88x923 uM). The lower affinity sites may arise from BK receptors which are not coupled to G-proteins. Membranes prepared from mock transfected COS-7 cells did not contain any detectable BK specific binding sites. This functional expression of a human BK bradykinin receptor is particularly suitable for designing methods of using the protein to screen for antagonists or agonists of this BK receptor site and, therefore, is critical for drug discovery in this important area.
Competition binding studies indicate that the cloned BK receptor binds BK analogues with the specificity of BK greater than lys-BK greater than met-lys-BK (Data not shown). In contrast, peptides reported to be specific for the BK-1 receptor have a very low affinity for this cloned receptor. At a concentration of 10 uM, the BK-1 agonist Des-Arg 9BK and the BK antagonist Des-Arg9, LeuBK inhibit BK binding by 18% and 11% respectively. No competition for BK binding is seen with the peptides angiotensin I and II, neurotensin, oxytocin, and endothelin. These results indicate that the receptor cloned and described in the instant application has the pharmacological properties expected for a BK-2 bradykinin receptor.
To further illustrate this principle, the ability of the human BK-2 receptor to interact with well known selective BK-2 antagonists is analyzed (Data not shown). Competition binding studies indicate that Hoe 140 (Hock et al., Br. J. Pharmacol. 102, 774-777 (1991)), D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]BK is a potent inhibitor with an IC50 for the cloned human receptor of 65 pM. This value is in sharp contrast to the IC50 of Hoe 140 previously reported from binding studies on guinea pig ileum membranes of 1.07 nM. See Hock et al. The higher affinity for Hoe 140 observed with the cloned human receptor of the instant invention may arise from structural differences between the human and guinea pig BK-2 receptors, from heterogeneity of guinea pig ileum BK-2 receptors, or from differences in experimental design. [3H]BK binding to the human BK-2 receptor of this invention may also be displaced by the known BK-2 antagonists D-Arg0-[Hyp2,3,-Thi5,8D-Phe7]BK, (IC50=27 nM) and [Thi5,8,D-Phe7]BK, (IC50=180 nM).
In order to simplify the following Examples and the Detailed Description, certain terms will be defined.
Fibroblasts are spindle shaped cells generally responsible for formation of extracellular fibers such as collagen. In this specification, a cDNA library was prepared from human CCD-16Lu fibroblast cells.
PCR is the polymerase chain reactionxe2x80x94a technique for copying the complementary strands of a target DNA molecule simaltaneously for a series of cycles until the desired amount is obtained. First, primers are synthesized that have nucleotide sequences complementary to the DNA that flanks the target region. The DNA is heated to separate the complementary strands and then cooled to let the primers bind to the flanking sequences. A heat-stable DNA polymerase is added, and the reaction is allowed to proceed for a series of replication cycles. Twenty will yield a millionfold amplification; thirty cycles will yield an amplification factor of one billion. See Appendix C, 1985, Saiki, Mullis et al.: Taq DNA polymerase.
Transfection is the incorporation by a cell of foreign DNA into cultured eucaryotic cells (such as COS cells) by exposing them to this DNA.
COS cells are a monkey cell line that has been transformed by an SV40 viral genome containing a defective origin of viral replication. When introduced into COS cells, recombinant RNAs or DNAs containing the SV40 origin and a foreign gene (such as BK-2 cDNA) replicate many copies.
SV40 is a DNA virus that readily infects cultured primate cells. SV40 replicates in the nuclei of host cells and becomes stably integrated into the host genome.
Plasmids are designated by a low case p preceded or followed by capital letters and/or numbers. The starting plasmids used in this invention are commercially available, are publicly available on an unrestricted basis, or can be constructed from such available plasmids in accordance with known procedures. In addition, other equivalent plasmids or constructs will be readily apparent to one skilled in the art. Vectors generally comprise plasmids, viruses (including phage), and integratable DNA fragments (i.e., fragments integratable into the host genome by recombination). Vectors may replicate and function independently of the host genome or may integrate into the host genome. Vectors are essentially replicable DNA constructs. Plasmids are the most commonly used form of vector. However, all other forms of vectors which serve an equivalent function of carrying or transporting a cDNA coding for a bradykinin BK-2 receptor are suitable for use herein.
An expression vector is a replicable DNA construct in which a DNA sequence encoding a BK-2 bradykinin receptor is operably linked to suitable control sequences capable of effecting the expression of BK-2 bradykinin receptor in a suitable host. Control sequences include a transcriptional promoter, an optional operator sequence to control trascription, a sequence encoding suitable mRNA binding sites, and sequences which control the termination of transcription and translation. Certain vectors, such as amplification vectors, do not need expression control domains but rather need the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.
Transformed host cells are cells which have been transformed or transfected with bradykinin BK-2 vectors constructed using recombinant DNA techniques. Expressed BK-2 bradykinin receptor will be deposited in the cell membrane of the host cell. Transformed cells may also be used for cloning or amplifying bradykinin BK-2 DNA.
Expression vectors normally contain a promoter which is recognized by the host cell. Viral sources often provide the transcriptional and translational control sequences necessary to transform vertebrate cells. Simian Virus 40 (SV40) is often used. As indicated earlier, COS cells contain a defective SV40 origin of replication and are subsequently transfected with an expression construct containing a bradykinin BK-2 cDNA and an active SV40 origin of replication. Alternatively, the host cell may provide an origin of replication if it is integrated into the host cell chromosome.
Cultures of cells derived from multicellular organisms are the preferred hosts for bradykinin BK-2 synthesis. Mammalian cells are the most preferred. Propagation of such cells in cell culture is known. See Kruse and Patterson, Ed. Tissue Culture, Academic Press (1973). Various mammalian cell lines include VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, BHK, COS-7, CV and MDCK cell lines. Preferrably, COS-7 cells or CHO cells are used in the instant invention. Expression vectors for these cells normally include an origin of replication, a promoter located upstream from the gene to be expressed, along with a ribosome binding site, RNA splice site (if intron-containing genomic DNA is used), a polyadenylation site, and a transcriptional termination sequence.
A transgenic mouse carrying the human BK-2 receptor gene may be generated by direct replacement of the mouse BK-2 receptor gene with the human BK-2 receptor gene by homologous recombination. The transgenic mouse carrying the human BK-2 gene will be useful in characterizing the in vivo efficacy of antagonists of the human BK-2 gene isolated from in vitro studies.
Compositions claimed in the instant invention may be prepared according to methods known in the art. For example, BK-2 bradykinin receptor protein may be mixed with pharmaceutically acceptable carriers. These carriers will be non-toxic to recipients or patients in need thereof at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining the BK receptor protein with buffers, antioxidants such as ascorbic acid, low molecular weight polypeptides, proteins, amino acids, carbohydrate including glucose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. BK receptors may be administered to counteract the effects of excess bradykinin or to absorb autoimmune anti-bradykinin BK-2 receptor antibody.